Multicellular development in the cellular slime mold Dictyostelium discoideum is regulated through cell-cell interactions that mediate signal transduction processes, resulting in cell-type differentiation an differential regulation of gene expression. This application is directed at understanding the mechanisms by which these interactions regulate the developmental switch that induces cell-type differentiation after the formation of the multicellular aggregate. The goal is to understand how the signaling processes initiated within the mound are involved in activating downstream transcriptional regulation. Some of this proposal concentrates on the continued analysis of the regulatory properties of the transcription factor GBF that is regulated by high levels of cAMP interacting with cell surface cAMP receptors. A combination of molecular, biochemical, and molecular genetic approaches will be used to dissect this pathway and understand how GBF mediates the induction of essentially all late genes whose expression is initiated at this time in development. In addition, the analysis of the pathway regulated by the lagC gene product, postulated to be a cell surface signaling molecule, will be pursued. How this pathway interacts with receptor-mediated pathways and GBF induction of gene expression will be examined. Analysis of cellular movement within the multicellular organism will be undertaken using time-lapse video microscopy to understand the abnormal spatial patterning in developmental mutants. The molecular mechanisms involved in activating the prespore differentiation pathway will be undertaken to understand the decision to differentiate into prespore versus prestalk cells. This question will initially be approached by purifying and cloning the genes encoding the transcription factors that are specific for inducing genes in prespore cells as they sort to differentiate. The recently developed REMI approach for random mutagenesis will be used to identify and then clone new gene products involved in mediating the onset of multicellular development and prespore cell differentiation. These will be coupled to other approaches, such as using the yeast two- hybrid system, to identify interacting gene products. The overall goal of this work is to understand at a molecular level the regulatory pathways that are involved in the decision-making process controlling multicellular development in this organism. Because of the relative simplicity of the system and the facility with which one can pursue multiple approaches to analyze gene function, It is expected that major inroads will be made into understanding not only how this organism differentiates but how the signaling pathways leads to changes in cell-type differentiation in eukaryotes in general. In addition, gene complementation approaches will be used to identity potential enhancers of regulatory pathways as well as high-copy-number suppressors to further expand the understanding of the mechanisms regulating these events. Together, these approaches should provide significant insight into how individual gene products interact to mediate differentiation.